Reid A. Roberts

Cutting Edge: NLRC5-Dependent Activation of the Inflammasome

The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1β and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function. RNA interference-mediated knockdown of NLRC5 nearly eliminated caspase 1, IL-1β, and IL-18 processing in response to bacterial infection, pathogen-associated molecular patterns, and damage-associated molecular patterns. This was confirmed in primary human monocytic cells. NLRC5, together with procaspase 1, pro–IL-1β, and the inflammasome adaptor ASC, reconstituted inflammasome activity that showed cooperativity with NLRP3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in a nucleotide-binding domain-dependent but leucine-rich repeat-inhibitory fashion. These results invoke a model in which NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome.

Nanoparticle clearance is governed by Th1/Th2 immunity and strain background

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol onto particles (PEGylation) extend circulation times; however, these particles are still cleared, and the processes involved in this clearance remain poorly understood. Here, we present an intravital microscopy-based assay for the quantification of nanoparticle clearance, allowing us to determine the effect of mouse strain and immune system function on particle clearance. We demonstrate that mouse strains that are prone to Th1 immune responses clear nanoparticles at a slower rate than Th2-prone mice. Using depletion strategies, we show that both granulocytes and macrophages participate in the enhanced clearance observed in Th2-prone mice. Macrophages isolated from Th1 strains took up fewer particles in vitro than macrophages from Th2 strains. Treating macrophages from Th1 strains with cytokines to differentiate them into M2 macrophages increased the amount of particle uptake. Conversely, treating macrophages from Th2 strains with cytokines to differentiate them into M1 macrophages decreased their particle uptake. Moreover, these results were confirmed in human monocyte-derived macrophages, suggesting that global immune regulation has a significant impact on nanoparticle clearance in humans.

Analysis of the Murine Immune Response to Pulmonary Delivery of Precisely Fabricated Nano- and Microscale Particles

Nanomedicine has the potential to transform clinical care in the 21st century. However, a precise understanding of how nanomaterial design parameters such as size, shape and composition affect the mammalian immune system is a prerequisite for the realization of nanomedicines translational promise. Herein, we make use of the recently developed Particle Replication in Non-wetting Template (PRINT) fabrication process to precisely fabricate particles across and the nano- and micro-scale with defined shapes and compositions to address the role of particle design parameters on the murine innate immune response in both in vitro and in vivo settings. We find that particles composed of either the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) or the biocompatible polymer polyethylene glycol (PEG) do not cause release of pro-inflammatory cytokines nor inflammasome activation in bone marrow-derived macrophages. When instilled into the lungs of mice, particle composition and size can augment the number and type of innate immune cells recruited to the lungs without triggering inflammatory responses as assayed by cytokine release and histopathology. Smaller particles (80×320 nm) are more readily taken up in vivo by monocytes and macrophages than larger particles (6 µm diameter), yet particles of all tested sizes remained in the lungs for up to 7 days without clearance or triggering of host immunity. These results suggest rational design of nanoparticle physical parameters can be used for sustained and localized delivery of therapeutics to the lungs.

Characterization of NLRP12 during the development of allergic airway disease in mice.

Among the 22 members of the nucleotide binding-domain, leucine rich repeat-containing (NLR) family, less than half have been functionally characterized. Of those that have been well studied, most form caspase-1 activating inflammasomes. NLRP12 is a unique NLR that has been shown to attenuate inflammatory pathways in biochemical assays and mediate the lymph node homing of activated skin dendritic cells in contact hypersensitivity responses. Since the mechanism between these two important observations remains elusive, we further evaluated the contribution of NLRP12 to organ specific adaptive immune responses by focusing on the lung, which, like skin, is exposed to both exogenous and endogenous inflammatory agents. In models of allergic airway inflammation induced by either acute ovalbumin (OVA) exposure or chronic house dust mite (HDM) antigen exposure, Nlrp12−/− mice displayed subtle differences in eosinophil and monocyte infiltration into the airways. However, the overall development of allergic airway disease and airway function was not significantly altered by NLRP12 deficiency. Together, the combined data suggest that NLRP12 does not play a vital role in regulating Th2 driven airway inflammation using common model systems that are physiologically relevant to human disease. Thus, the allergic airway inflammation models described here should be appropriate for subsequent studies that seek to decipher the contribution of NLRP12 in mediating the host response to agents associated with asthma exacerbation.

Analysis of human innate immune responses to PRINT fabricated nanoparticles with cross validation using a humanized mouse model.

Ideal nanoparticle (NP)-based drug and vaccine delivery vectors should be free of inherent cytotoxic or immunostimulatory properties. Therefore, determining baseline immune responses to nanomaterials is of utmost importance when designing human therapeutics. We characterized the response of human immune cells to hydrogel NPs fabricated using Particle Replication in Non-wetting Templates (PRINT) technology. We found preferential NP uptake by primary CD14(+) monocytes, which was significantly reduced upon PEGylation of the NP surface. Multiplex cytokine analysis of NP treated primary human peripheral blood mononuclear cells suggests that PRINT based hydrogel NPs do not evoke significant inflammatory responses nor induce cytotoxicity or complement activation. We furthered these studies using an in vivo humanized mouse model and similarly found preferential NP uptake by human CD14(+) monocytes without systemic inflammatory cytokine responses. These studies suggest that PRINT hydrogel particles form a desirable platform for vaccine and drug delivery as they neither induce inflammation nor toxicity. From the clinical editor: The authors here fabricated hydrogel nanorods using the PRINT (Particle Replication In Nonwetting Templates) fabrication process. They tested the interaction of human immune cells with these particles and found no immunoreactivity. This finding would suggest that monodisperse PRINT particles of identical shape and size could serve a variety of clinical applications.